目的探讨甲基转移酶5(methyltransferase-like 5,METTL5)在三阴乳腺癌(triple-negative breast cancer,TNBC)中的作用和潜在机制。方法采用免疫组织化学方法和Western blot检测TNBC肿瘤组织和细胞系中METTL5的表达情况。用靶向METTL5的s...目的探讨甲基转移酶5(methyltransferase-like 5,METTL5)在三阴乳腺癌(triple-negative breast cancer,TNBC)中的作用和潜在机制。方法采用免疫组织化学方法和Western blot检测TNBC肿瘤组织和细胞系中METTL5的表达情况。用靶向METTL5的shRNA(shRNA-METTL5)转染TNBC细胞后,用CCK-8、集落形成、伤口愈合以及Transwell实验分别检测细胞增殖活性、迁移与侵袭,Western blot检测Wnt/β-catenin信号关键蛋白的表达。构建异种移植瘤模型,验证敲降METTL5对TNBC细胞在体内生长以及Wnt/β-catenin信号活性的影响。结果METTL5在TNBC肿瘤组织和细胞系中表达上调(P<0.01)。敲降METTL5可抑制TNBC细胞的增殖、迁移和侵袭并降低了Wnt/β-catenin信号分子β-catenin、细胞周期蛋白(Cyclin)D1、基质金属蛋白酶(MMP)-2和MMP-7的表达(均P<0.01)。体内实验显示,敲降METTL5减缓了移植瘤生长和Wnt/β-catenin信号活性。结论敲降METTL5能抑制TNBC细胞的增殖、迁移与侵袭,其作用可能与抑制Wnt/β-catenin信号通路有关。展开更多
BACKGROUND Wnt/FZD-mediated signaling pathways are activated in more than 90%of hepatocellular carcinoma(HCC)cell lines.As a well-known secretory glycoprotein,Wnt3 can interact with FZD receptors on the cell surface,t...BACKGROUND Wnt/FZD-mediated signaling pathways are activated in more than 90%of hepatocellular carcinoma(HCC)cell lines.As a well-known secretory glycoprotein,Wnt3 can interact with FZD receptors on the cell surface,thereby activating the Wnt/β-catenin signaling pathway.However,the N-glycosylation modification site of Wnt3 and the effect of this modification on the biological function of the protein are still unclear.AIM To investigate the effect of Wnt3 N-glycosylation on the biological function of HCC cells.METHODS Site-directed mutagenesis was used to verify the Wnt3 N-glycosylation sites,actinomycin D treatment was used to detect the stability of Wnt3 after site-directed mutation,the binding of the N-glycosylation site-directed mutant Wnt3 to FZD7 was observed by laser confocal microscopy,and the effects of the N-glycosylation site-directed mutation of Wnt3 on the Wnt/β-catenin signaling pathway and the progression of HCC cells were detected by western blot and cell function experiments.RESULTS Wnt3 has two N-glycosylation-modified sites(Asn90 and Asn301);when a single site at amino acid 301 is mutated,the stability of Wnt3 is weakened;the binding ability of Wnt3 to FZD7 decreases when both sites are mutated simultaneously;and the level of proteins related to the Wnt/β-catenin signaling pathway is downregulated.Cell proliferation,migration and invasion are also weakened in the case of single 301 site and double-site mutations.CONCLUSION These results indicate that by inhibiting the N-glycosylation of Wnt3,the proliferation,migration,invasion and colony formation abilities of liver cancer cells can be weakened,which might provide new therapeutic strategies for clinical liver cancer in the future.展开更多
基金Supported by National Natural Science Foundation of China,No.81560390the Guizhou Medical University Cultivation Project of the National Natural Science Foundation of China,No.22NSFCP02Basic Research Project of Science and Technology Department of Guizhou Province,No.ZK[2024]General 136.
文摘BACKGROUND Wnt/FZD-mediated signaling pathways are activated in more than 90%of hepatocellular carcinoma(HCC)cell lines.As a well-known secretory glycoprotein,Wnt3 can interact with FZD receptors on the cell surface,thereby activating the Wnt/β-catenin signaling pathway.However,the N-glycosylation modification site of Wnt3 and the effect of this modification on the biological function of the protein are still unclear.AIM To investigate the effect of Wnt3 N-glycosylation on the biological function of HCC cells.METHODS Site-directed mutagenesis was used to verify the Wnt3 N-glycosylation sites,actinomycin D treatment was used to detect the stability of Wnt3 after site-directed mutation,the binding of the N-glycosylation site-directed mutant Wnt3 to FZD7 was observed by laser confocal microscopy,and the effects of the N-glycosylation site-directed mutation of Wnt3 on the Wnt/β-catenin signaling pathway and the progression of HCC cells were detected by western blot and cell function experiments.RESULTS Wnt3 has two N-glycosylation-modified sites(Asn90 and Asn301);when a single site at amino acid 301 is mutated,the stability of Wnt3 is weakened;the binding ability of Wnt3 to FZD7 decreases when both sites are mutated simultaneously;and the level of proteins related to the Wnt/β-catenin signaling pathway is downregulated.Cell proliferation,migration and invasion are also weakened in the case of single 301 site and double-site mutations.CONCLUSION These results indicate that by inhibiting the N-glycosylation of Wnt3,the proliferation,migration,invasion and colony formation abilities of liver cancer cells can be weakened,which might provide new therapeutic strategies for clinical liver cancer in the future.